Aromatic amidines as antiviral agents in animals

ABSTRACT

Novel [N,N-di(higher alkyl)aminomethyl]benzamidine and substituted compounds such as [N,N-di(higher alkyl)aminomethyl]-N-(2-propyl)-benzamidine and [N,N-di(higher alkyl)aminomethyl]-N-(p-hydroxyphenyl)-benzamidine and their non-toxic acid addition salts are useful for combating viral infections in vertebrate animals.

BACKGROUND OF THE INVENTION

Virus infections which attack animals, including man, are normallycontagious afflictions which are capable of causing great humansuffering and economic loss. Unfortunately, the discovery of antiviralcompounds is far more complicated and difficult than the discovery ofantibacterial and antifungal agents. This is due, in part, to the closestructural similarity of viruses and the structure of certain essentialcellular components such as ribonucleic and deoxyribonucleic acids.Nevertheless, numerous non-viral "antiviral agents", i.e. substances"which can produce either a protective or therapeutic effect to theclear detectable advantage of the virus infected host, or any materialthat can significantly enhance antibody formation, improve antibodyactivity, improve non-specific resistance, speed convalescence ordepress symptoms" [Herrman et al., Proc. Soc. Exptl. Biol. Med., 103,625 (1960)], have been described in the literature. The list of reportedantiviral agents includes, to name a few, interferon and syntheticmaterials such as amantadine hydrochloride, pyrimidines, biguanides,guanidine, pteridines and methisazone. Because of the rather narrowrange of viral infections that can be treated by each of the antiviralagents commercially available at the present time, new syntheticantiviral agents are always welcomed as potentially valuable additionsto the armamentarium of medical technology.

U.S. Pat. No. 3,906,044 discloses the antiviral activity of certainadamantyl amidine compounds of the formula: ##STR1## wherein n is 0 or1, and Ad is adamantyl or bridgehead carbon atom-substitutedalkyladamantyl. The antiviral activity of the compoundN-[bis-phenyl-(2-methoxy-5-chloro-phenyl)-methyl]-acetamidine isdisclosed in British Pat. No. 1,426,603.

SUMMARY OF THE INVENTION

It has now been found that certain novel benzamidine and N-substitutedbenzamidine compounds are capable of combating viral infections invertebrate animals. The novel compounds of this invention have theformula ##STR2## and the non-toxic acid addition salts thereof whereinR₁ and R₂ are each alkyl of from twelve to twenty-four carbon atoms; and

R₃ is selected from the group consisting of hydrogen; alkyl of from oneto six carbon atoms; alkenyl of from three to six carbon atoms;cycloalkyl of from three to eight carbon atoms; phenyl; phenylalkyl offrom seven to nine carbon atoms; pyridyl; pyrimidyl; dimethlamino;##STR3## --(CH₂)_(n) CH₂ OH, --(CH₂)_(n) SO₃ H, and --(CH₂)_(n) CF₃,wherein n is an integer of from one to six; and mono-- anddi-substituted phenyl wherein said substituents are selected from thegroup consisting of fluoro, chloro, bromo, hydroxyl, nitro,trifluoromethyl, alkyl and alkoxy of from one to three carbon atoms,dimethylamino, --N(CH₃)₃ ⁺ Cl⁻, --SO₂ NH₂, ##STR4## and --SO₂ R₄,wherein R₄ is alkyl of from one to three carbon atoms, provided thatwhen said phenyl ring is di-substituted at least one of saidsubstituents is selected from the group consisting of hydroxyl, alkyland alkoxy of from one to three carbon atoms, and dimethylamino.

The invention disclosed herein comprises the novel antiviral compoundsof formula I and the novel method of treating viral infections invertebrate animals characterized by administration of a pharmaceuticalcomposition containing an antivirally effective amount of a compound offormula I as the essential active ingredient.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I exhibit prophylactic antiviral activity invivo in vertebrate animals. It is probable that these compounds functionas antiviral agents by virtue of their ability to induce the productionof endogenous interferon, although the present invention is not to beconstrued as limited by such a theory.

By "non-toxic" acid addition salts is meant those salts which arenon-toxic at the dosages administered. The non-toxic acid addition saltswhich may be employed include such water-soluble and water-insolublesalts as the hydrochloride, dihydrochloride, hydrobromide, phosphate,diphosphate, nitrate, sulfate, acetate, hexafluorophosphate, citrate,gluconate, benzoate, propionate, butyrate, sulfosalicylate, maleate,laurate, malate, fumarate, succinate, oxalate, tartrate, amsonate(4,4'-diaminostilbene-2,2'-disulfonate), pamoate(1,1'-methylene-bis-2-hydroxy-3-naphthoate), stearate,3-hydroxy-2-naphthoate, p-toluenesulfonate, methanesulfonate, lactate,dilactate, and suramin salts.

One preferred group of the compounds of formula I consists of thehydrochloride, dihydrochloride, hydrobromide, dihydrobromide, phosphate,diphosphate, lactate, methanesulfonate and succinate salts of the basesof formula I.

Another preferred group of the compounds of formula I consists of thosewherein R₁ and R₂ are both normal alkyl.

Another preferred group of the compounds of formula I consists of thosewherein R₁ and R₂ are both normal alkyl and contain the same number ofcarbon atoms.

Another preferred group of the compounds of formula I consists of thosewherein R₁ and R₂ are both n-hexadecyl.

Another preferred group of the compounds of formula I consists of thosewherein R₁ and R₂ are both n-octadecyl.

Another preferred group of the compounds of formula I consists of thosewherein the benzene ring of said formula is meta-substituted.

The preferred substituents for R₃ are hydrogen; alkyl of from one tothree carbon atoms; allyl; phenylalkyl of from seven to nine carbonatoms; dimethylamino; ##STR5## --(CH₂)_(n) --SO₃ H and --(CH₂)_(n) CF₃,wherein n is an integer of from one to three; and para-mono-substitutedphenyl, wherein said substituent is selected from the group consistingof hydroxyl, methyl, methoxy, dimethylamino, --N(CH₃)₃ ⁺ Cl⁻ and --SO₂NH₂.

Particularly valuable are the following compounds:

m-[N,N-di(n-hexadecyl)aminomethyl]-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-(2-propyl)-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-(2,2,2-trifluoroethyl)-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-allyl-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-dimethylamino-benzamidine,

m-[N,N-di(hexadecyl)aminomethyl]-N-(p-hydroxyphenyl)-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-(p-methoxyphenyl)-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-methyl-benzamidine,

m-[N,N-di(n-hexadecyl)aminomethyl]-N-(p-dimethylaminophenyl)-benzamidine,and their non-toxic acid addition salts.

The compounds of this invention are prepared by methods familiar tothose skilled in the art. The first step is generally the condensationof the appropriate α-[N,N-di(higher alkyl)amino]-toluonitrile withethanthiol or ethanol in a hydrogen chloride saturated inert solventsuch as chloroform to form the corresponding ethylthio-benzimidate orethylbenzimidate dihydrochloride, as for example: ##STR6##

The second step is the reaction of H₂ NR₃ with the imidate. When R₃ isnot hydrogen and H₂ NR₃ is not strongly basic, the second step of thepreparation is the standard Pinner synthesis of amidines from imidates(Patai, S., ed., "The Chemistry of Amidines and Imidates", John Wileyand Sons, Inc., New York, 1975, pp. 283-341), i.e., the nucleophilicsubstitution of --NHR₃ for ethanthiol or ethanol in an inert solvent,e.g. chloroform, as for example: ##STR7## The reaction product is thedesired N-substituted benzamidine.

When R₃ is not hydrogen and H₂ NR₃ is strongly basic, use of thestandard synthesis described above yields the nitrile rather than theamidine. Efficient production of the amidine can be achieved, however,by pH control in the region 4-9 as e.g., with acetic acid or an aceticacid/sodium acetate buffer system as described in Examples 20-33.

Compounds of formula I wherein R₃ is hydrogen are prepared bycondensation of the appropriate α-[N,N-di(higheralkyl)amino]-toluonitrile with ethanol or ethanethiol in a hydrogenchloride saturated inert solvent such as dioxane to form thecorresponding ethylbenzimidate or ethylthiobenzimidate dihydrochloride,followed by nucleophilic substitution with NH₃ and elimination ofethanol or ethanethiol, which is carried out in ammonia saturatedethanol. The reaction product is the desired [N,N-di(higheralkyl)aminomethyl]benzamidine.

The compounds wherein R₃ is --(CH₂)_(n) SO₃ H, wherein n is an integerof from 1 to 6, are prepared by condensation of the appropriate[N,N-di(higher alkyl)aminomethyl] benzamidine (i.e. R₃ = H) with theappropriate γ-sultone or the appropriate sulfonic acid in an inertsolvent, e.g., 1,2-dichloroethane. The compounds wherein R₃ is ##STR8##are prepared in the same manner using oxalyl chloride and theappropriate benzamidine.

It is to be understood that any reaction-inert solvent may be used inplace of chloroform, dioxane or 1,2-dichloroethane in any of the methodsof preparation described above. The list of acceptable reaction solventsincludes, but is not limited to, chloroform, dioxane,1,2-dichloroethane, ethyl acetate and methylene chloride. Each of thereactions described above is typically performed at or near roomtemperature.

It is to be understood that any of the common procedures for amidinesynthesis referred to in the literature reviews, such as Patai, S., ed.,op. cit., arising from the appropriate intermediates such as imidates,thioimidates, iminoyl chlorides, thioamides, nitriles, amides oramidines, may be used to produce the compounds of this invention.

Acid addition salts of the bases of formula I may be prepared byconventional procedures such as by mixing the amidine compound in asuitable solvent with the required acid and recovering the salt byevaporation or by precipitation upon adding a non-solvent for the salt.Hydrochloride salts may readily be prepared by passing dry hydrogenchloride through a solution of the amidine compound in an organicsolvent.

The α-[N,N-di(higher alkyl)amino]-toluonitriles used as startingmaterials may be prepared by contacting α-bromotoluonitrile with anappropriate N,N-di(higher alkyl)amine in dimethylacetamide in thepresence of potassium carbonate. α-Bromotoluonitrile is an article ofcommerce obtainable, for example, from Shawnee Chemicals. TheN,N-di(higher alkyl)amine is obtained by refluxing a (higher alkyl)amine with the appropriate carboxylic acid in a suitable solvent such asxylene and then contacting the N-(higher alkyl)amide which is formedwith sodium bis(2-methoxyethoxy)-aluminum hydride in a suitable solventsuch as benzene to produce the desired N,N-di(higher alkyl)amine. Sodiumbis-(2-methoxy-ethoxy)aluminum hydride is an article of commerceobtainable, for example from Eastman Kodak Corporation as a 70% solutionin benzene under the trade name of Vitride. As will easily be recognizedby those skilled in the art, this procedure may be employed to prepareN,N-di(higher alkyl)amines in which the alkyl groups are eitheridentical or different. If an N,N-di(higher alkyl)amine with identicalalkyl groups is desired, a process comprising refluxing the mono-(higheralkyl)amine in a suitable solvent such as toluene in the presence ofRaney nickel catalyst to produce the desired N,N-di(higher akyl)aminemay also be employed. This latter process is not in common use becausetertiary amines, which are typically difficult to separate from thedesired secondary amine product, are frequently formed. This problem isnot serious, however, when the alkyl groups are higher alkyl (i.e.,twelve to twenty-four carbon atoms), because of the apparent sterichindrance to tertiary amine formation afforded by the great bulk of thealkyl moieties and the ease of separating tertiary from secondary(higher alkyl)amines.

The antiviral activity of the compounds of formula I is determined bythe following procedure. The test compound is administered to mice bythe intraperitoneal route eighteen to twenty-four hours prior tochallenging them with a lethal dose of encephalomyocarditis (EMC) virus.The survival rate is determined ten days after challenge and an ED₅₀[dosage level (mg of compound/kg body weight) required to obtain a fiftypercent survival rate] calculated. The procedure in which the drug isgiven eighteen to twenty-four hours before, and at a distinctlydifferent site from, virus injection is designed to eliminate localeffects between drug and virus and identify only those compounds whichproduce a systemic antiviral response.

Certain of the compounds of formula I were also tested for their abilityto induce circulating interferon in mice after parenteraladministration, using the procedure described by Hoffman, W. W. et al.,Antimicrobial Agents and Chemotherapy, 3, 498-501 (1973).

Parenteral, topical and intranasal administration of the above-describedamidines to an animal before exposure of the animal to an infectiousvirus provide rapid resistance to the virus. Such administration iseffective when given as much as five days prior to exposure to thevirus. Preferably, however, administration should take place from aboutthree days to about one day before exposure to the virus, although thiswill vary somewhat with the particular animal species and the particularinfectious virus.

When administered parenterally (subcutaneously, intramuscularly,intraperitoneally) the materials of this invention are used at a levelof from about 1 mg./kg. of body weight to about 250 mg./kg. body weight.The favored range is from about 5 mg./kg. to about 100 mg./kg. of bodyweight, and the preferred range from about 5 mg. to about 50 mg./kg. ofbody weight. The dosage, of course, is dependent upon the animal beingtreated and the particular amidine compound involved and is to bedetermined by the individual responsible for its administration.Generally, small doses will be administered initially with gradualincrease in dosage until the optimal dosage level is determined for theparticular subject under treatment.

Vehicles suitable for parenteral injection may be either aqueous such aswater, isotonic saline, isotonic dextrose, Ringer's solution, ornon-aqueous such as fatty oils of vegetable origin (cottonseed, peanutoil, corn, sesame) and other non-aqueous vehicles which will notinterfere with the efficacy of the preparation and are non-toxic in thevolume or proportion used (glycerol, ethanol, propylene glycol,sorbitol). Additionally, compositions suitable for extemporaneouspreparation of solutions prior to administration may advantageously bemade. Such compositions may include liquid diluents, for example,propylene glycol, diethyl carbonate, glycerol, sorbitol.

When the materials of this invention are administered, they are mosteasily and economically used in a dispersed form in an acceptablecarrier. When it is said that this material is dispersed, it means thatthe particles may be molecular in size and held in true solution in asuitable solvent or that the particles may be colloidal in size anddispersed through a liquid phase in the form of a suspension or anemulsion. The term "dispersed" also means that the particles may bemixed with and spread throughout a solid carrier so that the mixture isin the form of a powder or dust. This term is also meant to encompassmixtures which are suitable for use as sprays, including solutions,suspensions or emulsions of the agents of this invention.

In practicing the intranasal route of administration of this inventionany practical method can be used to contact the antiviral agent with therespiratory tract of the animal. Effective methods includeadministration of the agent by intranasal or nasopharyngeal drops and byinhalation as delivered by a nebulizer or an aerosol. Such methods ofadministration are of practical importance because they provide an easy,safe and efficient method of practicing this invention. For intranasaladministration of the agent, usually in an acceptable carrier, aconcentration of agent between 1.0 mg./ml. and 100 mg./ml. issatisfactory. Concentrations in the range of about 30 to 50 mg./ml.allow administration of a convenient volume of material.

For topical application the antiviral agents are most conveniently usedin an acceptable carrier to permit ease and control of application andbetter absorption. Here also concentrations in the range of from about1.0 mg./ml. to about 250 mg./ml. are satisfactory. In general, in theabove two methods of administration a dose within the range of about 1.0mg./kg. to about 250 mg./kg. of body weight and, preferably, from about5.0 mg./kg. to about 50 mg./kg. of body weight will be administered.

The compounds employed in this invention may be employed alone, i.e.,without other medicinals, as mixtures of more than one of theherein-described compounds, or in combination with other medicinalagents, such as analgesics, anesthetics, antiseptics, decongestants,antibiotics, vaccines, buffering agents and inorganic salts, to afforddesirable pharmacological properties. Further, they may be administeredin combination with hyaluronidase to avoid or, at least, to minimizelocal irritation and to increase the rate of absorption of the compound.Hyaluronidase levels of at least about 150 (U.S.P.) units are effectivein this respect although higher or lower levels can, of course, be used.

Those materials of this invention which are water-insoluble, includingthose which are of low and/or difficult solubility in water, are, foroptimum results, administered in formulations, e.g., suspensions,emulsions, which permit formation of particle sizes of less than about20μ. The particle sizes of the formulations influence their biologicalactivity apparently through better absorption of the active materials.In formulating these materials various surface active agents andprotective colloids are used. Suitable surface active agents are thepartial esters of common fatty acids, such as lauric, oleic, stearic,with hexitol anhydrides derived from sorbitol, and the polyoxyethylenederivatives of such ester products. Such products are sold under thetrademarks "Spans" and "Tweens," respectively, and are available fromICI United States Inc., Wilmington, Del. Cellulose ethers, especiallycellulose methyl ether (Methocel, available from the Dow Chemical Co.,Midland, Mich.) are highly efficient as protective colloids for use inemulsions containing the materials of this invention.

The water-soluble materials described herein are administered foroptimum results in aqueous solution. Typically they are administered inphosphate buffered saline. The water-insoluble compounds areadministered in formulations of the type described above or in variousother formulations as previously noted. Dimethylsulfoxide serves as asuitable vehicle for water-insoluble compounds. A representativeformulation for such compounds comprises formulating 25 to 100 mg. ofthe chosen drug as an emulsion by melting and mixing with equal parts ofpolysorbate 80 and glycerin to which hot (80° C.) water is added undervigorous mixing. Sodium chloride is added in a concentrated solution toa final concentration of 0.14 M and sodium phosphate, pH 7, is added toa final concentration of 0.01 M to give, for example, the followingrepresentative composition.

    ______________________________________                                                            mg./ml.                                                   ______________________________________                                        Drug                  50.0                                                    Polysorbate 80        50.0                                                    Glycerin              50.0                                                    Sodium Phosphate Monobasic Hydrous                                                                  1.4                                                     Sodium Chloride       7.9                                                     Water                 842.0                                                                         1001.3                                                  ______________________________________                                    

In certain instances, as where clumping of the drug particles occurs,sonication is employed to provide a homogeneous system.

The following examples illustrate the invention but are not to beconstrued as limiting the same.

EXAMPLE 1 Ethyl-m-[N,N-di(n-hexadecyl)aminomethyl]-benzimidateDihydrochloride

A mixture of α -[N,N-di(n-hexadecyl)amino]-m-toluonitrile (29.0 g., 0.05mole), ethanol (40 ml., 0.67 mole) and dioxane (100 ml.) was saturatedwith dry hydrogen chloride gas for 40 minutes at 15°-25° C. It was thenstoppered and held overnight at room temperature. Thin layerchromatography analysis (4:1, benzene:ethanol on silica gel) indicatedcomplete reaction of the nitrile. The mixture was evaporated in vacuoyielding the named product quantitatively as a foam [35.0 g., ˜ 100%yield, R_(f) 0.87 (4:1, benzene:ethanol on silica gel) ].

EXAMPLE 2 m-[N,N-Di-(n-hexadecyl)aminomethyl]-benzamidine

Ethyl-m-[N,N-di(n-hexadecyl)aminomethyl]-benzimidate dihydrochloride(35.0 g., 0.05 mole) was dissolved in ethanol (150 ml.) and the mixturesaturated with ammonia gas at 20° C. The mixture was held for threehours at 20° C., resaturated with ammonia gas at 20° C., and thenstoppered and held overnight at room temperature. The mixture wasevaporated in vacuo to a solid which was triturated with acetone (200ml.), filtered, washed with water (4 × 100 ml.), triturated again withacetone (2 × 100 ml.), filtered and dried in vacuo overnight [22.0 g.,74% yield, R_(f) 0.42 (4:1, benzene-ethanol on silicic acid)]. The crudeproduct was recrystallized from hot acetone (20.9 g., 70% yield,m.p.-forms a gel at 84° C.).

EXAMPLE 3 m-[N,N-Di(n-hexadecyl)aminomethyl]-N-(n-propane)sulfonicacid-benzamidine

m-[N,N-Di(n-hexadecyl)aminomethyl]-benzamidine (1.196 g., 2.0 mmoles)was added to a solution of 3-hydroxy-1-propane-sulfonic acid-γ-sultone(244 mg., 2.0 mmoles) dissolved in 1,2-dichloroethane (15 ml.). Themixture was held for 18 hours at room temperature. It was then dilutedto 300 ml. with ethyl acetate:ether (2:1), washed with 1N HCl (3 × 50ml.), washed with saturated aqueous sodium chloride solution (3 × 50ml.), dried (Na₂ SO₄) and evaporated in vacuo to an oil. The oil wascrystallized from 1,2-dimethoxyethane/acetonitrile [531 mg., 35% yield,R_(f) 0.35 (4:1, benzene:ethanol on silicic acid), m.p.-forms a gel at87°-95° C.].

EXAMPLE 4 m-[N,N-Di(n-hexadecyl)aminomethyl]-N-oxoaceticacid-benzamidine

In like manner to that described in Example 3 the compoundm-[N,N-di-(n-hexadecyl)aminomethyl]-N-oxoacetic acid-benzamidine wasprepared by using oxalyl chloride as starting material and a reactiontime of 1.5 hours. The oil was crystallized from 1,2-dimethoxyethane[34% yield, R_(f) 0.31 (4:1, benzene:ethanol on silicic acid),m.p.-forms a gel at 97°-105° C.].

EXAMPLE 5 Ethyl-m-[N,N-di(n-hexadecyl)aminomethyl]-thiobenzimidateDihydrochloride

A mixture of α-[N,N-di(n-hexadecyl)amino]-m-toluonitrile (23.2 g., 0.04mole), ethanthiol (6.0 ml., 0.08 mole) and chloroform (100 ml.) wassaturated with dry hydrogen chloride for 30 minutes at 20°-25° C. It wasthen stoppered and held for six days at 5° C. The mixture was evaporatedin vacuo to a foam which was crystallized by trituration with1,2-dimethoxyethane. The crude product was recrystallized from hot1,2-dimethoxyethane/chloroform [24.9 g., 88% yield, R_(f) 0.79 (4:1,benzene:ethanol on silicic acid), m.p. 109°-111° C.].

EXAMPLE 6m-[N,N-Di(n-hexadecyl)aminomethyl]-N-(p-methoxyphenyl)-benzamidine

A mixture of ethyl-m-[N,N-di(N-hexadecyl)aminomethyl] -thiobenzimidatedihydrochloride (1.074 g., 1.5 mmoles), p-anisidine (369 mg., 3.0mmoles) and chloroform (10 ml.) was held at room temperature for sixteenhours. It was then diluted to 400 ml. with chloroform, washed with 1NHCl (2 × 50 ml.), dried (Na₂ SO₄) and evaporated in vacuo to a foam. Thefoam was crystallized from 1,2-dimethoxyethane [868 mg., 73% yield,R_(f) 0.64 (4:1, benzene:ethanol on silicic acid), m.p.-forms a gel at84°-86° C.].

EXAMPLES 7-19

In like manner to that described in Example 6 the following compoundswere prepared by using appropriate reactants (H₂ N--R₃) in place ofp-anisidine: ##STR9##

    __________________________________________________________________________    Example            Reaction     Crystallization                               Number                                                                             R.sub.3       Time (hrs.)                                                                           Yield (%)                                                                          Solvent System.sup.a                                                                  M.P. (° C)                                                                   R.sub.f.sup.b                   __________________________________________________________________________     7                                                                                               16      88   DME      86-88.sup.d                                                                        .50                              8                                                                                  ##STR10##    16      88   DME     135-137                                                                             .47                              9                                                                                  ##STR11##    16      73   DME     164-167                                                                             .69                             10                                                                                  ##STR12##    16      67   DME     156-157                                                                             .73                             11                                                                                  ##STR13##    48      19   DME/CH.sub.3 CN                                                                        85-87.sup.d                                                                        .83                             12                                                                                  ##STR14##    48      59   DME/CHCl.sub.3                                                                        125.sup.d                                                                           .24                             13                                                                                  ##STR15##    48      62   DME     152-154                                                                             .76                             14                                                                                  ##STR16##     3      83   DME     167   .37                             15                                                                                  ##STR17##    48      68   DME      91-92.sup.d                                                                        .69                             16                                                                                  ##STR18##    48      89   DME/CHCl.sub.3                                                                        164.sup.d                                                                           .78                             17                                                                                  ##STR19##     36.sup.c                                                                              8   DME     128.sup.d                                                                           .70                             18                                                                                  ##STR20##    0.5     90   DME     167-169                                                                             .55                             19                                                                                  ##STR21##    48      15   Acetone  75.sup.d                                                                           .69                             __________________________________________________________________________     ##STR22##                                                                    .sup.b - 4:1, benzene:ethanol on silicic acid                                 .sup.c - reaction carried out at reflux                                       .sup.d - forms a gel                                                      

EXAMPLE 20 m-[N,N-Di(n-hexadecyl)aminomethyl]-N-cyclopentyl-benzamidine

Ethyl-m-[N,N-di(n-hexadecyl)aminomethyl]-thiobenzimidate dihydrochloride(1.074 g., 1.5 mmoles) was added to a solution of cyclopentylamine (255mg., 3.0 mmoles), glacial acetic acid (0.3 ml., 5.3 mmoles) andchloroform (10 ml.). The mixture was held for 72 hours at roomtemperature. It was then diluted to 300 ml. with chloroform, washed withsaturated aqueous sodium bicarbonate solution (3 × 50 ml.), washed withsaturated aqueous sodium chloride solution (3 × 50 ml.), dried (Na₂ SO₄)and filtered. The filtrate was acidified with a 10% solution ofanhydrous hydrogen chloride in dioxane (5 ml.) and then evaporated invacuo to an oil. The oil was crystallized from warm 1,2-dimethoxyethane[850 mg., 77% yield, R_(f) 0.30 (4:1, benzene:ethanol on silicic acid),m.p.-forms a gel at 78° C.].

EXAMPLES 21-27

In like manner to that described in Example 20 the following compoundswere prepared by using appropriate reactants (H₂ N--R₃) in place ofcyclopentylamine: ##STR23##

    __________________________________________________________________________    Example        Reaction     Crystallization                                   Number                                                                             R.sub.3   Time (hrs.)                                                                          Yield (%)                                                                           Solvent System.sup.a                                                                  M.P. (° C)                                                                   R.sub.f.sup.b                       __________________________________________________________________________    21                                                                                           48     28    DME     172-176                                                                             .75                                 22                                                                                  ##STR24##                                                                              1.5    88    DME     154-157                                                                             .37                                 23   N(CH.sub.3).sub.2                                                                       16     23    DME/CH.sub.3 CN                                                                       82.sup.d                                                                            .38                                 24   CH.sub.2 CHCH.sub.2                                                                     16     77    DME/CH.sub.3 CN                                                                       70.sup.d                                                                            .29                                 25   CH(CH.sub.3).sub.2                                                                      48     55    DME     95.sup.d                                                                            .30                                 26   CH.sub.2 CH.sub.3.sup.c                                                                 72     84    DME     91.sup.d                                                                            .22                                 27   CH.sub.3.sup.c                                                                          72     90    DME     106.sup.d                                                                           .18                                 __________________________________________________________________________     ##STR25##                                                                    .sup.b - 4:1, benzene:ethanol on silicic acid                                 .sup.c - ethylamine (methylamine)bubbled as a gas into acetic                 acid:chloroform solution                                                      .sup.d - forms a gel                                                      

EXAMPLE 28m-[N,N-Di(n-hexadecyl)aminomethyl]-N-(2,2,2-trifluoroethyl)-benzamidine

Ethyl-m-[N,N-di(n-hexadecyl)aminomethyl]-thiobenzimidate dihydrochloride(1.074 g., 1.5 mmoles) was added to a slurry of2,2,2-trifluoroethylamine hydrochloride (406 mg., 3.0 mmoles) andanhydrous sodium acetate (246 mmoles) in chloroform (10 ml.) and glacialacetic acid (0.3 ml., 5.3 mmoles). The mixture was held for 12 hours atroom temperature. It was then diluted to 300 ml. with chloroform, washedwith saturated aqueous sodium bicarbonate solution (2 × 50 ml.), washedwith saturated aqueous sodium chloride solution (2 × 50 ml.), dried (Na₂SO₄) and filtered. The filtrate was acidified with a 10% solution ofanhydrous hydrogen chloride in dioxane (5 ml.) and then evaporated invacuo to a foam. The foam was crystallized from 1,2-dimethoxyethane [974mg., 86% yield, R_(f) 0.39 (4:1, benzene:ethanol on silicic acid),m.p.-forms a gel at 125°-127° C.].

EXAMPLES 29-33

In like manner to that described in Example 28 the following compoundswere prepared by using appropriate reactants (H₂ N--R₃ ·HCl) in place of2,2,2-trifluoroethylamine hydrochloride: ##STR26##

    __________________________________________________________________________    Example            Reaction     Crystallization                               Number                                                                             R.sub.3       Time (hrs.)                                                                          Yield (%)                                                                           Solvent System.sup.a                                                                  M.P. (° C)                                                                   R.sub.f.sup.b                   __________________________________________________________________________    29                                                                                               24     57    DME      77-79.sup.c                                                                        .43                             30                                                                                  ##STR27##    48     60    DME/CH.sub.3 CN                                                                       115-118.sup.c                                                                       .46                             31                                                                                  ##STR28##    16     29    DME     238.sup.c                                                                           .60                             32                                                                                  ##STR29##     3     55    DME/CHCl.sub.3                                                                        163-166                                                                             .00                             33                                                                                  ##STR30##    24     40    DME/water                                                                             140.sup.c                                                                           .27                             __________________________________________________________________________     ##STR31##                                                                    .sup.b - 4:1, benzene:ethanol on silicic acid                                 .sup.c - forms a gel                                                      

EXAMPLES 34-35

In like manner to that described in Examples 3-4 the following compoundsmay be prepared by using appropriate reactants in place of3-hydroxy-1-propanesulfonic acid-γ-sultone:

    ______________________________________                                         ##STR32##                                                                    Example                                                                       Number  R.sub.3      Reactant                                                 ______________________________________                                        34      CH.sub.2 SO.sub.3 H                                                                        iodosulfonic acid                                        35      (CH.sub.2).sub.6 SO.sub.3 H                                                                6-hydroxy-1-hexanesulfonic                                                    acid-ζ-sultone                                      ______________________________________                                    

EXAMPLES 36-74

In like manner to that described in Examples 6-19 the followingcompounds may be prepared by using appropriate reactants (H₂ N--R₃) inplace of p-anisidine: ##STR33##

    ______________________________________                                        Example                                                                       Number         R.sub.5                                                        ______________________________________                                        36             2-fluoro                                                       37             3-fluoro                                                       38             2-chloro                                                       39             3-chloro                                                       40             2-bromo                                                        41             3-bromo                                                        42             4-bromo                                                        43             2-hydroxyl                                                     44             3-hydroxyl                                                     45             2-nitro                                                        46             4-nitro                                                        47             2-trifluoromethyl                                              48             3-trifluoromethyl                                              49             4-trifluoromethyl                                              50             2-methyl                                                       51             3-methyl                                                       52             2-n-propyl                                                     53             3-ethyl                                                        54             4-isopropyl                                                    55             2-methoxy                                                      56             3-methoxy                                                      57             2-n-propyloxy                                                  58             3-ethoxy                                                       59             4-isopropyloxy                                                 60             2-dimethylamino                                                61             3-dimethylamino                                                62             2-SO.sub.2 NH.sub.2                                            63             3-SO.sub.2 NH.sub.2                                            64             2-(CO)OCH.sub.3                                                65             3-(CO)OCH.sub.3                                                66             4-(CO)OCH.sub.3                                                67             2-(CO)O(CH.sub.2).sub.2 CH.sub.3                               68             3-(CO)OCH.sub.2 CH.sub.3                                       69             4-(CO)OCH(CH.sub.3).sub.2                                      70             2-SO.sub.2 CH.sub.3                                            71             3-SO.sub.2 CH.sub.3                                            72             2-SO.sub.2 CH(CH.sub.3).sub.2                                  73             3-SO.sub.2 (CH.sub.2).sub.2 CH.sub.3                           74             4-SO.sub.2 CH.sub.2 CH.sub.3                                   ______________________________________                                    

EXAMPLES 75-92

In like manner to that described in Examples 6-19 the followingcompounds may be prepared by using appropriate reactants (H₂ N--R₃) inplace of p-ansidine: ##STR34##

    ______________________________________                                        Example                                                                       Number         R.sub.6        R.sub.7                                         ______________________________________                                        75       chloro         methyl                                                76       trifluoromethyl                                                                              methoxy                                               77       bromo          dimethylamino                                         78       chloro         dimethylamino                                         79       methyl         methyl                                                80       methyl         methoxy                                               81       2-propyl       dimethylamino                                         82       methoxy        dimethylamino                                         83       1-propyloxy    --SO.sub.2 (CH.sub.2).sub.2 CH.sub.3                  84       ethyl          --SO.sub.2 NH.sub.2                                   85       ethoxy         --(CO)OCH(CH.sub.3).sub.2                             86       1-propyl       --(CO)OCH.sub.3                                       87       dimethylamino  dimethylamino                                         88       2-propyloxy    dimethylamino                                         89       dimethylamino  --SO.sub.2 NH.sub.2                                   90       dimethylamino  --SO.sub.2 CH.sub.3                                   91       dimethylamino  --(CO)OCH.sub.3                                       92       dimethylamino  --(CO)O(CH.sub.2).sub.2 CH.sub.3                      ______________________________________                                    

EXAMPLES 93-98

In like manner to that described in Examples 20-27 the followingcompounds may be prepared by using appropriate reactants (H₂ N--R₃) inplace of cyclopentylamine: ##STR35##

    ______________________________________                                        Example                                                                       Number               R.sub.3                                                  ______________________________________                                        93             n-hexyl                                                        94             2-butenyl                                                      95             2-hexenyl                                                      96             cyclopropyl                                                    97             cyclooctyl                                                     98             phenyl (1-propyl)                                              ______________________________________                                    

EXAMPLES 99-103

In like manner to that described in Examples 28-33 the followingcompounds may be prepared by using appropriate reactants (H₂ NR₃ ·HCl)in place of 2,2,2-trifluoroethylamine hydrochloride:

    ______________________________________                                         ##STR36##                                                                    Example                                                                       Number       R.sub.3                                                          ______________________________________                                         99          (CH.sub.2).sub.2 OH                                              100          (CH.sub.2).sub.7 OH                                              101          (CH.sub.2).sub.6 CF.sub.3                                        102                                                                                         ##STR37##                                                       103                                                                                         ##STR38##                                                       ______________________________________                                    

EXAMPLES 104-112

In like manner to that described in Examples 28-33 the followingcompounds may be prepared by using appropriate reactants (H₂ NR₃ ·HCl)in place of 2,2,2-trifluoroethylamine hydrochloride:

    ______________________________________                                         ##STR39##                                                                    Example                                                                       Number     R.sub.6        R.sub.7                                             ______________________________________                                        104        hydroxyl       chloro                                              105        hydroxyl       methoxy                                             106        hydroxyl       dimethylamino                                       107        hydroxyl       SO.sub.2 NH.sub.2                                   108        hydroxyl       (CO)OCH.sub.3                                       109        hydroxyl       N(CH.sub.3).sub.3 .sup.+Cl.sup.-                    110        N(CH.sub.3).sub.3 .sup.+Cl.sup.-                                                             methyl                                              111        N(CH.sub.3).sub.3 .sup.+Cl.sup.-                                                             dimethylamino                                       112        hydroxyl       ethyl                                               ______________________________________                                    

EXAMPLE 113 Antiviral Activity ofm-[N,N-di(n-hexadecyl)aminomethyl]-N-allylbenzamidine Dihydrochloride

Three groups of ten female albino mice (20-25 g. body weight) were givensingle 0.5 ml. intraperitoneal injections containing dosage levels of1.5, 5, and 15 mg. of the named compound/kg. body weight, respectively.A fourth control group was given no such injection. Eighteen totwenty-four hours later all four groups were challenged with a 0.2 ml.subcutaneous injection containing 20-30 times the LD₅₀, the dosage levelcausing a 50% death rate in ten days, of encephalomyocarditis (EMC)virus. The following survival data were recorded for the following tendays:

    ______________________________________                                        Dosage Level                                                                  of Named Number of Survivors on Day Number                                     Compound                                                                              0     1     2   3   4   5   6   7   8   9   10                                                    S.sub.r                                          ______________________________________                                        15 mg./kg.                                                                             10    10    10  10   9  8   8   8   8   8   8                                                     80                                                                            5 10 10 10 10 10 6 6 6 5 5 5 53                                               1.5 10 10 10 10  9 5 4 2 2 2 1 19                                             0 (control) 10 10 10  9  3 1 1 0 0 0 0 --        ______________________________________                                    

Antiviral activity is expressed as the relative survival (S_(r)) inexperimental groups compared to the controls on the tenth day afterchallenge. S_(r) is defined by the formula ##EQU1## wherein S_(r) =relative survival

S_(x) = percent survival after ten days in experimental group

x_(i) = number of survivors on the ith day in experimental group

e_(i) = number of survivors on the ith day in control group

The ED₅₀ [dosage level (mg. of compound/kg. body weight) required toobtain a fifty percent survival rate] is determined graphically byplotting S_(r) (ordinate) vs. 1n dosage level (abscissa) and thenfitting the points with a line of predetermined slope by least squares.The dosage level at which this fitted line has an ordinate of 50 isequivalent to the ED₅₀.

This graphical method was used to determine an ED₅₀ for the namedcompound of 4.7 mg. (as dihydrochloride salt)/kg.

EXAMPLES 114-143

In like manner to that described in Example 113 the antiviral activitywas determined for the compounds listed below.

    ______________________________________                                        Example  Compound Prepared in                                                 Number   Example Number   ED.sub.50 (mg./kg.).sup.a                           ______________________________________                                        114      2                4.7                                                 115      3                8.0                                                 116      4                9.9                                                 117      6                5.3                                                 118      7                12.3                                                119      8                16.0                                                120      9                7.7                                                 121      10               49.3                                                122      11               35.7                                                123      12               4.9                                                 124      13               21.6                                                125      14               8.0                                                 126      15               38.0                                                127      16               7.0                                                 128      17               12.9                                                129      18               17.9                                                130      19               8.9                                                 131      20               27.3                                                132      21               47.5                                                133      22               7.8                                                 134      23               5.0                                                 135      25               2.8                                                 136      26               7.6                                                 137      27               5.7                                                 138      28               3.8                                                 139      29               6.9                                                 140      30               7.7                                                 141      31               11.7                                                142      32               7.4                                                 143      33               37.2                                                ______________________________________                                         .sup.a all as mg. dihydrochloride salt except for Example 115 (mg. free       base)                                                                    

EXAMPLE 144 Ability ofm-[N,N-di(n-hexadecyl)aminomethyl]-N-(p-hydroxyphenyl)-benzamidine toInduce Circulating Interferon

A quantity of the named compound was fused with equal weights ofpolysorbate 80 and glycerol. The mixture was then homogenized in hot0.14 M NaCl containing 0.01 sodium phosphate, pH 7 (PBS). The resultingoil-in-water emulsion was readily diluted with PBS.

Female Swiss mice (20-25 g. body weight) were injected (intraperitoneal)with an amount of the above diluted emulsion containing 25 mg. of thenamed compound/kg. body weight. Eight, twelve, sixteen and twenty hoursafter injection samples of plasma were withdrawn from the mice. Thesesamples were then serially diluted. L-929 mouse fibroblasts wereincubated on microtiter plates with aliquots of the various samples ofserially diluted plasma for eighteen hours at 37° C. The fibroblastmonolayers were then washed with protein-free medium and challenged with10-40 times the TCID₅₀, the dose in which 50% of the cultures areinfected, of vesicular stomatitis virus (VSV). The virus was allowed toabsorb for one hour at 37° C. before addition of 0.2 ml. of maintenancemedium. The cultures were scored and analyzed about twenty-four toforty-eight hours later and the plasma interferon level, the reciprocalof the plasma dilution at which fifty percent of the cultures areprotected, determined. The following data were obtained.

    ______________________________________                                        Plasma Interferon Levels (units/ml.)                                          Time (hrs.) after Injection                                                   8        12          16          20                                           ______________________________________                                        102      276         143         76                                           ______________________________________                                    

EXAMPLES 145-150

In like manner to that described in Example 144 the ability to inducecirculating interferon was determined for the compounds listed below.

    ______________________________________                                                            Plasma Interferon Levels (units/ml.)                      Ex.   Compound Prepared                                                                           Time (hrs.) after Injection                               No.   in Example Number                                                                           8       12    16    20                                    ______________________________________                                        145    2            76      116    56   48                                    146   24            26       60   110   102                                   147   25            <17     114    34   154                                   148   27            37       95   100   71                                    149   28            38      160   126   49                                    150   30            66       87    61   64                                    ______________________________________                                    

EXAMPLE 151

Compounds wherein R₁ and R₂ are not both n-(hexadecyl) and/or the phenylring of formula I is not meta-substituted may be prepared in like manneras described in Examples 1-33 for the correspondingm-[N,N-di(n-hexadecyl)] compounds by using the appropriate startingmaterials, and tested for antiviral activity in like manner as describedin Example 113.

What is claimed is:
 1. A compound of the structure ##STR40## and thenon-toxic acid addition salts thereof wherein R₁ and R₂ are each alkylof from twelve to twenty-four carbon atoms; andR₃ is selected from thegroup consisting of hydrogen; alkyl of from one to six carbon atoms;alkenyl of from three to six carbon atoms; cycloalkyl of from three toeight carbon atoms; phenyl; phenylalkyl of from seven to nine carbonatoms; ##STR41## and mono- and di-substituted phenyl wherein saidsubstituents are selected from the group consisting of fluoro, chloro,bromo, hydroxyl, nitro, trifluoromethyl, alkyl and alkoxy of from one tothree carbon atoms, dimethylamino, --N(CH₃)₃ ⁺ Cl⁻, --SO₂ NH₂, ##STR42##and --SO₂ R₄, wherein R₄ is selected from the group consisting of alkylof from one to three carbon atoms, provided that when said phenyl ringis di-substituted at least one of said substituents is selected from thegroup consisting of hydroxyl, alkyl and alkoxy of from one to threecarbon atoms, and dimethylamino.
 2. A compound of claim 1 wherein R₁ andR₂ are normal alkyl.
 3. A compound of claim 2 wherein R₁ and R₂ have anequal number of carbon atoms.
 4. A compound of claim 1 wherein R₁ and R₂are n-hexadecyl.
 5. A compound of claim 1 wherein R₁ and R₂ aren-octadecyl.
 6. A compound of claim 1 wherein the benzene ring of saidstructure is meta substituted.
 7. A compound of claim 1 wherein R₁ andR₂ are n-hexadecyl and the benzene ring of said structure is metasubstituted.
 8. A compound of claim 1 wherein R₃ is selected from thegroup consisting of hydrogen; alkyl of from one to three carbon atoms;allyl; phenylalkyl of from seven to nine carbon atoms; ##STR43## andpara-mono-substituted phenyl, wherein said substituent is selected fromthe group consisting of hydroxyl, methyl, methoxy, dimethylamino,--N(CH₃)₃ ⁺ Cl⁻ and --SO₂ NH₂.
 9. A compound of claim 7 wherein R₃ ishydrogen.
 10. A compound of claim 7 wherein R₃ is allyl.
 11. A compoundof claim 7 wherein R₃ is 2-propyl.
 12. A compound of claim 7 wherein R₃is p-hydroxyphenyl.
 13. A compound of claim 7 wherein R₃ isp-dimethylaminophenyl.